PROJECT SUMMARY The goal of this study is to develop a targeted, injectable plasmid delivery system that is capable of efficient direct reprogramming of cardiac fibroblasts to cardiomyocytes (CMCs) in vivo, aiding in cardiac tissue regeneration following a myocardial infarction (MI) or ?heart attack?. Cardiovascular disease (CVD) is the leading cause of death worldwide and resulted in 17.9 million deaths in 2016 alone. Cardiac fibrosis, a major contributor to CVD is which results from traumatic injury such as MI. A promising treatment avenue is direct reprogramming of cardiac fibroblasts into CMCs to regenerate the myocardial tissue and regain functional post-infarct heart tissue. Several recent studies have demonstrated that a combination of transcription factors, Gata4, Mef2c, and Tbx5 delivered simultaneously can directly reprogram cardiac fibroblasts to CMCs and induce CMC phenotype in vitro and in vivo. Unfortunately, while cardiac fibroblast to CMC differentiation is now a possibility, implementation of this approach as a CVD treatment has not been viable. The gene delivery vehicles (polyplexes) developed in the Ma Lab have been shown to drive bone tissue regeneration in vivo, setting an important precedent on which this study is based. Preliminary data using an improved polyplex has thus far driven in vitro reprogramming of cardiac fibroblasts into CMCs, a promising proof-of-concept result. In the proposed study, researchers will be further improving the recently developed system by improving the gene expression through nuclear targeting using a Nuclear Localization Signal (NLS). Preliminary results indicate this improves gene expression compared to polyplexes alone. Further, researchers will be driving direct reprogramming in vivo using a newly developed hydrogel scaffold to implement sustained delivery of the polyplexes. The hypothesis is that through addition of a NLS and sustained delivery of plasmid DNA using an injectable hydrogel, the nuclear localization and amount of plasmid delivered will be increased, resulting in an increase in in vivo reprogramming of fibroblasts into CMCs. This will be evaluated using PCR and immunohistochemistry to compare gene expression and later MRI to evaluate functional changes in the heart. This research aims to establish the first regenerative treatment option for patients who have suffered a heart attack. This work may likely lay the framework for an effective non-viral gene delivery system for use in treatment of other lost or diseased tissues, especially the fibrosis of other tissue/organ systems.